The proteome, the dynamic sum total of proteins expressed by the genome of a given cell, contains complex networks of regulatory pathways responsible for growth, metabolism, and response to external stimuli. Analysis of the subcellular localization of the proteome is critical for understanding these processes protein-protein interactions occur within subcellular compartments, and the translocation of sets of protein within the cell represents a measurable outcome of the activation of a given regulatory pathway. The budding yeast Saccharomyces cerevisiae provides a eukaryotic model system amenable to large-scale study of protein Iocalization. The aims of the study presented here are 1) to generate a comprehensive subcellular localization map of the yeast proteome through the construction and analysis of a green fluorescent protein (GFP)-tagged yeast library. 2) To elucidate and identify features of the proteome as a function of subcellular localization, including evaluation and complementation of current models of protein localization and protein-protein interactions. 3) To apply the GFP-tagged library as a tool for the analysis of a proteome-scale biological question, specifically the identification, from within the subset of nuclear proteins identified in the study, the targets of individual nuclear importins. Evolutionary conservation of proteins involved in many basic cellular functions makes the data from this study broadly applicable to a wide spectrum of eukaryotic species, from yeast to humans.